我们感谢安东尼J. Koleske，博士提供YFP的小鼠。

第1部分：微光学显微镜设置<ol><li>我们使用的microprisms 1毫米，直角棱镜，从Optosigma公司（图1）购买的BK7玻璃制成的。 Microprisms处理尽可能使用镊子。 </li><li>斜边的微涂有增强银提供大于97％的反射率从400 - 2000纳米。这提供了内部反射的激光激发光下发出荧光。 </li><li> Microprisms总是在可能时使用产钳处理。手套戴在任何时候都保持棱镜清洁。 </li><li>我们使用一个定制的双光子显微镜的小动物成像能力。动物，立体设备，被放置在一个3轴机动阶段。 </li><li>该显微镜是连接到Windows PC运行ScanImage软件。 ScanImage是图像采集软件在Matlab的书面Pologruto 等 1 。 </li><li>微成像，我们收集分辨率1024 × 1024或512 x 512像素的图像。此外，我们通常在2毫秒/线或4毫秒/线扫描。 </li><li>实验中使用两种类型的目标是： <ul><li>较低的数值孔径（NA）为宽视场的视图成像空中目标（ 见试剂和仪器表） 。 </li><li> NA与玻璃能够最大限度地减少由0引起的球面像差校正领一个更高的空中目标- 2毫米的玻璃（见试剂和仪器表）。 </li></ul></li><li>当动物准备一个低倍率目标下成像，对准激光与微顶部的方形光栅扫描模式。这将有助于东方的形象和激光功率的使用效率。 </li><li>使用玻璃校正领一个更高的NA目标，设置校正领正确的约1毫米的玻璃。一旦在实验过程中出现了荧光图像，可以仔细显微镜内达到优化的校正领，以最大限度地提高成像分辨率。 </li></ol>第2部分：非生存的动物手术和微插入<ol><li>整个过程的长度，从最初的切口，直至完成成像，取决于实验的目标。然而，可以预料，非生存的外科手术和微安置约30 - 40分钟才能完成。 </li><li>小鼠（P30 - P60），称重，麻醉，适当使用注射氯胺酮（100毫克/公斤）和甲苯噻嗪（10毫克/公斤）的IP适合外科手术的一个平面。 </li><li>在整个实验中，动物应检查每次15-20分钟，以评估其麻醉水平。如果动物的反应，一个坚定的脚趾捏，氯胺酮/甲苯​​噻嗪的补充剂量是必需的。通常情况下，补充的剂量初始剂量的三分之一，并可以立即腹腔注射的注射器提前做好准备，如果需要的话。 </li><li>一旦鼠标是适当的麻醉，大多数动物的头的头发剃掉使用＃40刀片的微调。 </li><li>奈尔®应用到余下的头发在头上，以帮助创建一个表面的头发可能在成像方式的微。 5分钟后，奈尔®是使用棉签拭子清除。 </li><li>麻醉的动物是正确放入鼠标立体设备。动物的外科手术和成像会议期间保持在一个充满水的加热垫设置在36摄氏度。 </li><li>妥善固定在动物的头，一个干净的切口，使用下来的头骨内侧线手术刀分开皮肤。 </li><li>小金额的3％的双氧水是放在一个棉签拭子和应用扫清之间的头骨和皮肤膜的头骨。 </li><li>过氧化氢也有助于揭示前囟，知道从何处执行的开颅手术，插入微的一个重要里程碑。 </li><li>开颅手术开始前，耳酒吧和咬栏适当的调整，倾斜等，它基本上是与表级暴露颅骨。这提供了一个更好的平台，为开颅。此外，这使得传入激发光垂直棱镜的顶面。这将有助于最大限度地减少光学像差，同时成像。 </li><li>一个小的牙科毛刺（〜0.8毫米头的大小）是连接到一个DREMEL ®工具，使用一个灵活的延伸臂。 craniotomies，我们设置了约7000至10000 rpm的速度。 </li><li>牙科毛刺脱脂沿着头骨，直到建立一个方形的凹槽是在骨。广场两侧约2-3毫米，长度为中心的1.5毫米的尾鳍和1毫米，横向到前囟。 </li><li>持续变薄，直到一个小口是通过颅骨骨。一对钳CAñ解除远离颅骨骨瓣。 </li><li>硬脑膜可分为皮质中的微前需要删除。最好控制出血，让血液凝固几分钟表面。随后，凝固的血是使用手术前插入微海绵删除。 </li><li>为了帮助协助与皮质棱镜的对齐方式，垂直的微面是一字排开平行镊子处理。 </li><li>有了正确举行棱镜，整个微棱镜顶部插入，直到与新皮层的表面平齐。 </li><li>正确插入时，微应保持在适当的位置。任何出血，结果是最小的，可以用一个小的手术海绵吸收。一旦棱镜动物成像准备。 </li><li>微已经在动物使用后，它可以多次使用。但是，它需要得到适当的清洗，以消除任何剩余的生物材料。这可以通过一个由浸成甲醇，盐酸的体积小，浸入棱镜。清洗微然后可以在镜头纸包裹，直到将来使用。 </li></ol>第3部分：代表结果<img src="/files/ftp_upload/1509/1509fig1.jpg" alt="图1" /> 图1：正确的角度微的图像。本实验所用的棱镜是一毫米，BK7玻璃棱镜加强斜边允许内部的激发和发射光的反射银涂层。 <img src="/files/ftp_upload/1509/1509fig2.jpg" alt="图2" /> 图2：V层YFP的锥体神经元的图像。宽视场的成像与微显示一个人口大锥体细胞体〜800 - 900微米深的皮质表面。此外，扩大对层的顶树突我也可以得到解决。比例尺= 200微米。 <img src="/files/ftp_upload/1509/1509fig3.jpg" alt="图3" /> 图3：提供足够的空间分辨率来解决V层锥体神经元顶树突树突棘与微一起使用的一个更高的数值孔径目标。比例尺= 10微米。 <img src="/files/ftp_upload/1509/1509fig4.jpg" alt="图4" /> 图4：使用微尾静脉注射和荧光染料得到的图像。图像显示垂直，大口径的船只从脑深部地区和分支通过皮质体积较小的毛细血管延伸。比例尺= 200微米。 <img src="/files/ftp_upload/1509/1509fig5.jpg" alt="图5" /> 图5：使用较高的数值孔径目标，新皮层中的毛细血管网络，可以更详细的影像。比例尺= 50微米。 <img src="/files/ftp_upload/1509/1509fig6.jpg" alt="图6" /> 图6：Microprisms也允许成像红血细胞（红细胞）通过船只流动。不吸收荧光染料的红细胞，因此作为整个船只的暗条纹。整个船只的线扫描，可以得到红细胞的流量和速度的测量。比例尺= 10微米（在顶部和底部的形象单杠），250毫秒（在底部图像竖线）。

<ol>
	<li>Pologruto, T. A., Sabatini, B. L., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ScanImage:+flexible+software+for+operating+laser+scanning+microscopes.">ScanImage: flexible software for operating laser scanning microscopes.</a> Biomed Eng Online. 2, 13-13 (2003).</li><li>Feng, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+neuronal+subsets+in+transgenic+mice+expressing+multiple+spectral+variants+of+GFP.">Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP.</a> Neuron. 28 (1), 41-41 (2000).</li><li>Kleinfeld, D., Mitra, P. P., Helmchen, F., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluctuations+and+stimulus-induced+changes+in+blood+flow+observed+in+individual+capillaries+in+layers+2+through+4+of+rat+neocortex.">Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex.</a> Proc Natl Acad Sci U S A. 95 (26), 15741-15741 (1998).</li></ol>

动物实验是由美国耶鲁大学的IACUC规定的准则和法规的规定执行。

使用微成像实验相对来说比较简单，在大脑皮层的体内研究提供了许多优点。使用这种技术的有代表性的例子包括在V层皮层神经元的表达黄色荧光蛋白的转基因小鼠拍摄的图像。使用microprisms，人们可以看到一个大V层的锥体细胞机构的收集，近1毫米以下皮质表面。也看到了延长塔夫茨大学（图2）前分歧，通过所有的表面层的顶树突。用于这些实验的老鼠是转基因动物表达YFP的，只有在V层神经元...

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<td>With enhanced silver reflective coating-(R77)</td>
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in vivo imaging of deep cortical layers using a microprism

我们现在活体成像的皮层组织，使用了深刻的脑成像探头在一个微形的协议。 Microprisms 1毫米大小，有一个斜边的反射涂层，让内部的激发和发射光的反射。微探针同时图像多个角度皮质层通常仅见于片的筹备工作。图片是一个大型的收集领域的视图（〜900微米）。此外，我们提供了生存​​的非外科手术的细节和显微镜设置。代表性的结果，包括从你的YFP - H - 1小鼠显示他们的顶树突延长通过表面的皮质层，并延伸到塔夫茨V层锥体神经元的图像。决议足以形象V层神经元的胞体附近的树突棘。一个荧光染料的尾静脉注射，揭示了在皮层血管的复杂网络。流经毛细血管的红细胞（红血球）线扫描揭示RBC速度和通量率可以得到。这种新颖的微探针是一个优雅的，但功能强大的可视化深层细胞的结构和在体内的皮质功能的新方法。

Watch this Scientific Journal Video about In vivo Imaging of Deep Cortical Layers using a Microprism at JoVE.com

In vivo Imaging of Deep Cortical Layers using a Microprism

直角microprisms允许插入到小鼠大脑皮层多个皮质层的深成像与通常在切片中发现了一个观点。一毫米microprisms提供了一个广阔的领域的视图（〜900微米）和空间分辨率足以解决树突棘。我们证明V层神经成像和新皮层的血管成像，使用microprisms。

在使用微深皮质层的活体成像

We present a protocol for in vivo imaging of cortical tissue using a deep-brain imaging probe in the shape of a microprism.  Microprisms are 1-mm in size ...

in-vivo-imaging-of-deep-cortical-layers-using-a-microprism

Research

JoVE Journal

Biology

In vivo Imaging of Deep Cortical Layers using a Microprism

11.3K 次观看.  Yale University. 直角microprisms允许插入到小鼠大脑皮层多个皮质层的深成像与通常在切片中发现了一个观点。一毫米microprisms提供了一个广阔的领域的视图（〜900微米）和空间分辨率足以解决树突棘。我们证明V层神经成像和新皮层的血管成像，使用microprisms。

视频: In vivo Imaging of Deep Cortical Layers using a Microprism

Preparation of Dissociated Mouse Cortical Neuron Cultures

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures can be used for a variety of applications including immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging, protein and/or RNA isolation. These cultures also provide a platform to study the neuronal development of transgenic animals that carry a late embryonic or postnatal lethal gene mutation. The procedure is relatively straight forward, requires some experience in tissue culture technique and should not take longer than two to three hours if you are properly prepared. Careful separation of the cortical rind from the thalamo-cortical fiber tract will reduce the number of unwanted non-neuronal cells. To increase yields of neuronal cells triturate the pieces of the cortical tissue gently after the enzyme incubation step. This is imperative as it prevents unnecessary injury to cells and premature neuronal cell death. Since these cultures are maintained in the absence of glia feeder cells, they also offer an added advantage of growing cultures enriched in neurons.

This video shows a procedure for generating neuronal cultures from late embryo and early postnatal mouse cortex. These cultures can be used for immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging and provide a platform to study the neuronal development of transgenic animals that carry a postnatal lethal gene mutation.

游离的小鼠皮层神经元文化的制备

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures ...

科研

生物学

Calcium Imaging of Cortical Neurons using Fura-2 AM

Calcium imaging is a common technique that is useful for measuring calcium signals in cultured cells. Calcium imaging techniques take advantage of calcium indicator dyes, which are BAPTA-based organic molecules that change their spectral properties in response to the binding of Ca2+ ions. Calcium indicator dyes fall into two categories, ratio-metric dyes like Fura-2 and Indo-1 and single-wavelength dyes like Fluo-4. Ratio-metric dyes change either their excitation or their emission spectra in response to calcium, allowing the concentration of intracellular calcium to be determined from the ratio of fluorescence emission or excitation at distinct wavelengths.  The main advantage of using ratio-metric dyes over single wavelength probes is that the ratio signal is independent of the dye concentration, illumination intensity, and optical path length allowing the concentration of intracellular calcium to be determined independently of these artifacts. One of the most common calcium indicators is Fura-2, which has an emission peak at 505 nM and changes its excitation peak from 340 nm to 380 nm in response to calcium binding.  Here we describe the use of Fura-2 to measure intracellular calcium elevations in neurons and other excitable cells.

Calcium signals play a key role in many cellular processes including gene expression, survival and differentiation. Here we demonstrate how to perform calcium imaging using Fura-2 AM. Calcium imaging is a valuable tool to study the regulation of intracellular calcium in real time and its regulation of signaling cascades.

使用FURA - 2上午皮层神经元的钙成像

Calcium imaging is a common technique that is useful for measuring calcium signals in cultured cells. Calcium imaging techniques take advantage of calcium ...

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

4T1 mouse mammary tumor cells can be implanted sub-cutaneously in nu/nu mice to form palpable tumors in 15 to 20 days. This xenograft tumor model system is valuable for the pre-clinical in vivo evaluation of putative antitumor compounds.
The 4T1 cell line has been engineered to constitutively express the firefly luciferase gene (luc2). When mice carrying 4T1-luc2 tumors are injected with Luciferin the tumors emit a visual light signal that can be monitored using a sensitive optical imaging system like the IVIS Spectrum. The photon flux from the tumor is proportional to the number of light emitting cells and the signal can be measured to monitor tumor growth and development. IVIS is calibrated to enable absolute quantitation of the bioluminescent signal and longitudinal studies can be performed over many months and over several orders of signal magnitude without compromising the quantitative result.
Tumor growth can be monitored for several days by bioluminescence before the tumor size becomes palpable or measurable by traditional physical means. This rapid monitoring can provide insight into early events in tumor development or lead to shorter experimental procedures.
Tumor cell death and necrosis due to hypoxia or drug treatment is indicated early by a reduction in the bioluminescent signal. This cell death might not be accompanied by a reduction in tumor size as measured by physical means. The ability to see early events in tumor necrosis has significant impact on the selection and development of therapeutic agents.
Quantitative imaging of tumor growth using IVIS provides precise quantitation and accelerates the experimental process to generate results.

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

Mammary tumor cells expressing luciferase are implanted subcutaneously in mice and visualized using optical imaging to monitor tumor growth and development non-invasively in a longitudinal study.

使用IVIS频谱的乳腺肿瘤在体内生物发光成像

4T1 mouse mammary tumor cells can be implanted sub-cutaneously in nu/nu mice to form palpable tumors in 15 to 20 days. This xenograft tumor model system ...

In-vivo Centrifugation of Drosophila Embryos

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in buoyant density. However, when cells are disrupted prior to centrifugation, proteins and organelles in this non-native environment often inappropriately stick to each other. Here we describe a method to separate organelles by density in intact, living Drosophila embryos. Early embryos before cellularization are harvested from population cages, and their outer egg shells are removed by treatment with 50% bleach. Embryos are then transferred to a small agar plate and inserted, posterior end first, into small vertical holes in the agar. The plates containing embedded embryos are centrifuged for 30 min at 3000g. The agar supports the embryos and keeps them in a defined orientation. Afterwards, the embryos are dug out of the agar with a blunt needle. Centrifugation separates major organelles into distinct layers, a stratification easily visible by bright-field microscopy. A number of fluorescent markers are available to confirm successful stratification in living embryos. Proteins associated with certain organelles will be enriched in a particular layer, demonstrating colocalization. Individual layers can be recovered for biochemical analysis or transplantation into donor eggs. This technique is applicable for organelle separation in other large cells, including the eggs and oocytes of diverse species.

In-vivo Centrifugation of Drosophila Embryos

We describe a method to separate organelles by density in living Drosophila embryos. Embryos are embedded in agar and centrifuged. This technique yields reproducible separation of major organelles along the anterior-posterior embryo axis. This method facilitates colocalization experiments and yields organelle fractions for biochemical analysis and transplantation experiments.

果蝇胚胎的体内离心

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in ...

Imaging Protein-protein Interactions in vivo

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using F&ouml;rster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface.

Imaging Protein-protein Interactions in vivo

This protocol describes how to image protein-protein interactions using a FRET-based proximity assay.

成像蛋白质 - 蛋白质相互作用在体内</em

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method makes it possible to count the numbers of protein molecules produced in one cell during sequential, five-minute time windows. It requires a fluorescence microscope with laser excitation power density of ~0.5 to 1 kW/cm2, which is sufficiently sensitive to detect single fluorescent protein molecules in live cells. The fluorescent reporter used in this method consists of three parts: a membrane targeting sequence, a fast-maturing, yellow fluorescent protein and a protease recognition sequence. The reporter is translationally fused to the N-terminus of a protein of interest. Cells are grown on a temperature-controlled microscope stage. Every five minutes, fluorescent molecules within cells are imaged (and later counted by analyzing fluorescence images) and subsequently photobleached so that only newly translated proteins are counted in the next measurement.
Fluorescence images resulting from this method can be analyzed by detecting fluorescent spots in each image, assigning them to individual cells and then assigning cells to cell lineages. The number of proteins produced within a time window in a given cell is calculated by dividing the integrated fluorescence intensity of spots by the average intensity of single fluorescent molecules. We used this method to measure expression levels in the range of 0-45 molecules in single 5 min time windows. This method enabled us to measure noise in the expression of the &lambda; repressor CI, and has many other potential applications in systems biology.

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the &lambda; repressor CI 1.

单分子成像的基因调控<em在体内</em使用转位激活裂解（CoTrAC）

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within tissues. Many semi-quantitative systems have been developed for scoring expression using immunohistochemistry, but inherent subjectivity limits reproducibility and accuracy of results. Furthermore, the investigation of spatially overlapping biomarkers such as nuclear transcription factors is difficult with current immunohistochemistry techniques. We have developed and optimized a system for simultaneous investigation of multiple proteins using high throughput methods of multiplexed immunohistochemistry and multispectral imaging. Multiplexed immunohistochemistry is performed by sequential application of primary antibodies with secondary antibodies conjugated to horseradish peroxidase or alkaline phosphatase. Different chromogens are used to detect each protein of interest. Stained slides are loaded into an automated slide scanner and a protocol is created for automated image acquisition. A spectral library is created by staining a set of slides with a single chromogen on each. A subset of representative stained images are imported into multispectral imaging software and an algorithm for distinguishing tissue type is created by defining tissue compartments on images. Subcellular compartments are segmented by using hematoxylin counterstain and adjusting the intrinsic algorithm. Thresholding is applied to determine positivity and protein co-localization. The final algorithm is then applied to the entire set of tissues. Resulting data allows the user to evaluate protein expression based on tissue type (ex. epithelia vs. stroma) and subcellular compartment (nucleus vs. cytoplasm vs. plasma membrane). Co-localization analysis allows for investigation of double-positive, double-negative, and single-positive cell types. Combining multispectral imaging with multiplexed immunohistochemistry and automated image acquisition is an objective, high-throughput method for investigation of biomarkers within tissues.

Immunohistochemistry is a powerful lab technique for evaluating protein localization and expression within tissues. Current semi-automated methods for quantitation introduce subjectivity and often create irreproducible results. Herein, we describe methods for multiplexed immunohistochemistry and objective quantitation of protein expression and co-localization using multispectral imaging.

蛋白表达和共定位使用复用免疫组织化学染色和多光谱成像定量

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

测量的刚度<em&gt;离体</em&gt;鼠标主动脉用原子力显微镜

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy (OCM)

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical coherence microscopy (OCM) has been demonstrated to be capable of imaging small animal hearts with high spatial resolution and ultrahigh imaging speed. The high image contrast and noninvasive properties make OCM ideal for performing longitudinal studies without requiring tissue dissections or staining. Drosophila has been widely used as a model organism in cardiac developmental studies due to its high number of orthologous human disease genes, its similarity of molecular mechanisms and genetic pathways with vertebrates, its short life cycle, and its low culture cost. Here, the experimental protocols are described for the preparation of Drosophila and optical imaging of the heartbeat with a custom OCM system throughout the life cycle of the specimen. By following the steps provided in this report, transverse M-mode and 3D OCM images can be acquired to conduct longitudinal studies of the Drosophila cardiac morphology and function. The en face and axial sectional OCM images and the heart rate (HR) and cardiac activity period (CAP) histograms, were also shown to analyze the heart structural changes and to quantify the heart dynamics during Drosophila metamorphosis, combined with the videos constructed with M-mode images to trace cardiac activity intuitively. Due to the genetic similarity between Drosophila and vertebrates, longitudinal study of heart morphology and dynamics in fruit flies could help reveal the origins of human heart diseases. The protocol here would provide an effective method to perform a wide range of studies to understand the mechanisms of cardiac diseases in humans.

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy OCM

Here, the experimental protocols are described for preparing Drosophila at different developmental stages and performing longitudinal optical imaging of Drosophila heartbeats using a custom optical coherence microscopy (OCM) system. The cardiac morphological and dynamical changes can be quantitatively characterized by analyzing the heart structural and functional parameters from OCM images.

果蝇的制备及心功能体内使用光学相干显微镜成像纵向（OCM）

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical ...